Since the 1975 report by Kohler and Milstein regarding cell fusion technology [Nature, 256, 495 (1975)], the monoclonal antibody has been very useful in various fields, including basic research into cell physiology and immunology, detection, quantification, separation and purification of biological trace components, and diagnosis and treatment of various diseases. Thanks to advances in gene recombination technology, diagnostically or therapeutically more useful antibodies, such as mouse/human chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies and toxin fusion antibodies have recently been prepared [Encyclopedia of Human Biology, 5, 81 (1991)]. Mouse/human chimeric antibodies and humanized antibodies, in particular, are advantageous for human administration, because the constant region and/or frame region of the mouse antibody, which is responsible for most immunogenicity, is of the human type [Nature, 314, 268 (1985); Nature, 321, 522 (1986)].
In expressing such recombinant antibodies, animal cells, especially mouse myeloma cells and CHO cells, are normally used. However, the use of these cells is economically problematic due to some drawbacks, such as expensive culture medium and long cultivating time. Also, since the degree of recombinant protein expression in animal cells is very low, various attempts have been made to achieve high expression. However, large amounts of medium must be used to obtain practically sufficient amounts of antibody, or cultivating time has to be extended much more with application of feed [Bio/Technology, 10, 169 (1992)], because the amount of protein expressed per unit volume of medium is generally low. Another drawback resides in the process for isolating the desired antibody; a great deal of time and labor is required to purify the protein from the culture broth. As for antibody expression in Escherichia coli, which is expected to offer high expression, many attempts have been reported, but most have failed to achieve practically acceptable levels of expression. The reasons for this situation include the following: 1) It is very difficult to express the entire IgG antibody molecule in Escherichia coli, due to complex conformation, especially S-S crosslinkages. 2) Although high expression has been achieved for antibodies of low molecular weight, such as functional antigen binding fragments Fv, Fab and Fab' [Science, 240, 1038 (1988); Nature, 347, 497 (1990); Bio/Technology, 10, 163 (1992)], single-chain antibodies (scAb) [Science, 242, 423 (1988)] and single-domain antibodies (dAb) [Nature,. 341, 544 (1989)], inactive inclusion bodies often form; active forms have rarely been recovered in sufficient amounts. 3) In Escherichia coli, the expressed antibody accumulates intracellularly in insoluble form, its purification taking vast amounts of time and labor, since the desired antibody must be isolated from the mixture of proteins derived from Escherichia coli after disruption of the cells of Escherichia coli. Furthermore, the desired antibody is liable to be decomposed by proteases derived from the host, Escherichia coli, during the complicated process. In short, it is difficult to produce an antibody in active form at high purity in large amounts by conventional methods.
As stated above, when a gene product of complex conformation from a higher organism is produced in a bacterial host-vector system such as Escherichia coli, it often accumulates in cells in the form of an inactive inclusion body [Journal of Biotechnology, 1, 307 (1984)] or undergoes decomposition [Gene, 34, 1 (1985)]; recovery is difficult while bioactivity is maintained.
The importance of recombinant antibodies, which are expected to exhibit various bioactivities, has steadily increased in the fields of diagnosis and treatment; there is strong need for a method of producing an antibody in an active form at low cost in large amounts.
Some bacteria of the genus Bacillus are known to produce certain extracellular protein; for example, Bacillus brevis is reported as possessing such function [Methods in Enzymology, 217, 23 (1993)]. By testing such Bacillus brevis bacteria for high production of proteins and no or little extracellular secretion of proteases, several strains have been selected and cloned with prominent characteristics. For example, Udaka [Agric. Biol. Chem., 40, 523(1976)] and Takagi [Agric. Biol. Chem.., 53, 691(1989)] established the strains of Bacillus brevis 47 and HPD31, respectively. Using these strains as host, with vectors containing DNA which encodes the promoter and signal peptides of extracellular enzyme gene or cell wall protein gene, for example of bacterial origin, .alpha.-amylases derived from Bacillus licheniforis [J. Bacteriol., 169, 1239(1987)] or derived from Bacillus stearothermophilus [Agric. Biol. Chem., 53, 2279(1989)] and .beta.-amylase derived from Clostridium thermosulfurogenes have been expressed at such production amounts of 0.5-3 g/L and 1.6 g/L, respectively.
Although many attempts to express proteins of eukaryote origin, from fungi to mammalians, in expression system of Bacillus brevis have been tried, human epithelial growth factor (EGF) of animal origin [Proc. Natl. Acad. Sci. USA, 86, 3589 (1989)], which consists of 53 amino acid sequences and has a relatively simple structure with small molecular size, is a solitary instance of high expression at such production amounts of 0.24-1.1 g/L. Taka-amylase A derived from Aspergillus oryzae [Journal of the Agricultural Chemical Society of Japan, 64, 728 (1990)], porcine pepsinogen [Appl. Microbiol. Biotechnol., 34, 297 (1990)], human salivary .alpha.-amylase (H. Konishi; Master Thesis in Nagoya University) and human interleukin-2 (Y. Takimura; Master Thesis in Nagoya University) of eukaryote origin have been also reported to be expressed in the Bacillus brevis expression system, the production amounts are very low, at such production amounts of 0.01-0.06 g/L. Moreover, physiologically-active proteins, such as antibodies, of mammalian origin with high molecular weights and complicated structures, have never been reported to be produced at high amounts in the Bacillus brevis expression system.